Human respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are cytoplasmic enveloped RNA viruses of the family Pneumoviridae. Their genomes are single strands of negative-sense RNA of 15.2 kb (RSV) or 13.3 kb (HMPV) that encode 10 mRNAs and 11 unique proteins (RSV) or 8 mRNAs and 9 unique proteins (HMPV). Each virus encodes a nucleoprotein N, phosphoprotein P, matrix protein M, small hydrophobic protein SH, major glycoprotein G, fusion glycoprotein F, polymerase factors M2-1 and M2-2, and the polymerase protein L. In addition, RSV encodes two nonstructural proteins NS1 and NS2. Murine pneumonia virus (MPV, previously known as pneumonia virus of mice, PVM) is a close relative of RSV whose natural host is the mouse and which provides a convenient permissive animal model. We presently are developing new attenuated RSV strains as vaccine candidates, but this work is in progress and not ready for presentation. In the present report, we describe a basic research project. We performed (in collaboration with NCATS) a genome-wide high-throughput siRNA screen of more than 20,000 cellular genes to identify cellular proteins involved in RSV infection. Human airway epithelial A549 cells were engineered to express Ds-Red as a viability marker, and were infected with RSV expressing green fluorescent protein (GFP) as a marker for viral gene expression. Three siRNAs per cellular gene were evaluated individually for reduction in GFP expression as a measure of inhibition of RSV infection. Computer analysis identified and corrected for siRNA off-target effects. The top 155 hits (i.e., the greatest reductions in GFP without excessive loss of viability) were re-evaluated with three additional siRNAs each, and the top 65 hits were identified. Ingenuity Pathway Analysis of the primary 155 selected candidate genes indicated that integrin signaling was one of the canonical pathways most associated with RSV infection. Among the top 65 genes identified for further analysis, four genes were associated with integrin signaling: actin-related protein 2, ARP2; ADP-ribosylation factor 1, ARF1; integrin alpha-V, ITGAV; and mitogen-activated protein kinase 3, MAPK3. We focused first on actin-related protein 2 (ARP2). ARP2 knockdown by specific siRNA did not reduce RSV entry or gene expression during the first 24 h of infection, but decreased viral gene expression thereafter. This late effect that appeared to be due to inhibition of viral spread to neighboring cells. Consistent with reduced spread, there was a 10-fold reduction in the release of infectious progeny virions in cells in which ARP2 expression was knocked down with specific siRNA. In addition, RSV infection was found to greatly stimulate the formation of filopodia, which are long slender projections from the cell surface. Progeny virus were associated with the filopodia, and contact between these RSV-carrying filopodia and neighboring cells appeared to deliver virus to these neighbors. In addition, RSV infection resulted in increased cell motility in A549 cells, and this motility appeared to be another way in which to deliver progeny RSV to neighboring cells. SiRNA-mediated knockdown of ARP2 expression strongly reduced filopodia formation and motility, indicating a role for ARP2 in both processes. Expression of the RSV F protein alone by a plasmid or heterologous viral vector induced filopodia, indicating a new role for the RSV F protein in RSV infection, specifically in inducing filopodia formation and virus spread. Thus, this study identified roles for ARP2, RSV F protein, and filopodia in RSV production and cell-to-cell spread. In other experiments, we used siRNAs to knock down expression of the other three integrin pathway candidates noted above, namely ARF1, ITGAV, and MAPK3. Knockdown of each of these three proteins reduced the production of progeny RSV, and the effect was increased if ARP2 was simultaneously knocked down. Two of the proteins, namely ARF1 and ITGAV, appeared reduce the efficiency of RSV entry based on siRNA-mediated knock down experiments. Further studies of the underlying mechanism are currently under way. These experiments highlight cellular proteins and cellular pathways that are involved in the various stages of RSV infection and replication.